FISSION YEAST WITH DNA-POLYMERASE DELTA TEMPERATURE-SENSITIVE ALLELES EXHIBITS CELL-DIVISION CYCLE PHENOTYPE

DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (polalpha+ and poldelta+) are essential for cell viability. Disruption of either the polalpha+ or poldelta+ gene results in distinct terminal phenotypes. The S.pombe poldelta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three poldelta conditional lethal alleles. We replaced the wild type chromosomal copy of poldelta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted poldelta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional poldelta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.